Number of rat ova implanting after sub-sterilizing X-irradiation of one or both ovaries.

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Number of R a t Ova Implanting after Sub-sterilizing
X-irradiation of One or Both Ovaries'
ROBERT H. KREHBIEL AND JAMES C. PLAGGE
Department of Anatomy, University of Illinois, College of Medicine,
Chicago, Illinois
Irradiation with €4001 of x-ray was applied to the exteriorized left
ovary, to both ovaries or to the left ovary after unilateral (right) castration of 60 day
old rats. Treated females were placed with the male at 60, 70, 80 or 90 days of age.
Females were explored at eight or nine days after sperm were found in the morning smears to determine the number of implantations.
The number of matings was reduced during the period of normal vaginal cycles.
Matings did occur after the unset of continuous vaginal cornification.
Animals with an irradiated left and a normal right ovary maintained an average
number of implantations through pregnancies one to seven. The normal ovary comsensated for the loss of production by the irradiated ovary. Bilateral irradiation
caused a decrease in implantations in the second and third pregnancies. In individual
cases, however, the normal complement for a single ovary occurred as late as 201 days
after irradiation. The irradiated ovary in the absence of the other ovary compensated
by producing more implantations; its period of production was limited to 31 days
following irradiation.
ABSTRACT
Large doses of x-ray to the rat ovary
cause changes in the estrous cycle, and in
time, produce an infertile animal. Mandl
('59a) noted occasional mating by the rat
after a sterilizing dose of x-ray. These
matings did not produce pregnancy although pseudopregnancy did occur. Ingram ('59), using smaller doses of x-ray,
obtained pregnancies and concluded that
the length of the fertile period thereafter
is inversely associated with the dosage.
He also suggested that the decrease in fertility was due to a reduction of available
ova. Similar findings have been reported
by others (Drips and Ford, '32; Rugh and
Wolff, '57 and Nuzhdin et al., '55). All
of these observations were made using
animals in which the entire ovarian tissue
was exposed to irradiation. Pencharz and
Long ('32) irradiated only one ovary in
the rat and found that subsequent pregnancies were produced and maintained by
the untreated ovary. Krehbiel and Plagge
('62), on the other hand, observed implantations on the irradiated side after
unilateral exposure to x-ray. Therefore, in
the present investigation, both bilateral
and unilateral irradiation of the ovary
with a sub-sterilizing dose (800 r ) were
undertaken in an attempt to clarify the
effects on fertility.
MATERIAL AND METHODS
With the animal in a protective lead
shield, the ovaries were exteriorized and
irradiated with 800 r at 60 days of age in
the manner described previously (Plagge
and Krehbiel, '62). The left ovary of 22
rats and both ovaries of 22 rats were irradiated. The third group of 12 rats had the
left ovary irradiated and the right ovary
removed.
Vaginal smears were obtained beginning two weeks prior to irradiation and
were continued through the period of observation. Some females were placed with
the male the evening of the day of irradition. Others were placed with the male
for the first time 10, 20 or 30 days later.
The number of implantations was determined by exploring the female eight or
nine days after sperm were found in the
morning smear. If pregant, the animal
was isolated until she delivered. The young
were examined superficially for abnormalities and sacrificed. In some cases the
female was returned to the male cage
during the postpartum period. Cycles and
mating were observed up to 300 days following irradiation.
3
Aided by grants from National Institutes of Health,
United States Public Health Service and the University
of Illinois Research Board, The Graduate College.
257
258
ROBERT H . KREHBIEL AND JAMES C . PLAGGE
OBSERVATIONS AND DISCUSSION
Vaginal cycles in all animals continued
more or less regularly for two months
following irradiation. During this time, in
animals exposed to the male, there were
periods of vaginal cornification in which
no sperm were found. During the early
post-irradiation period (1-60 days) sperm
were seen 59 times in 127 observations of
vaginal cornification in females with an
irradiated and an untreated ovary. Comparable results were obtained when both
ovaries were irradiated (41 in 11 1 smears).
In unilaterally castrated and irradiated females, sperm were found in 27 of 60
cycles. These ratios may indicate a lack
of coordination of vaginal and ovarian
activity, as reported by Mandl ('59b), or
perhaps, a failure of the ovaries to produce
a normal psychic estrus consistently.
Matings occurred during the period of
regular cycling and also after the onset of
continuous vaginal cornification. No definite pattern was seen in the freqeuncy of
the later matings. In a few cases, the females accepted the male for periods of
three or four days. There also was a suggestion that these animals were mating
at cyclic intervals (fig. 1; no. 1,034 and
1,048).
The untreated right ovary in unilaterally
irradiated females usually insured pregnancy (table 1). Following each of the
50 fertile matings, implantations were
found on the non-irradiated side whereas
they occurred in only 26 uteri of the
irradiated side. When both ovaries were
irradiated there was a marked drop in
fertile matings. However, many of the 98
negative matings occurred after irregular
cycles or continuous cornification had
started. In females which were unilaterally castrated and irradiated the infertility
Fig. 1 Typical vaginal smear records of female rats receiving 800 r of x-ray to the left ovary
and the right ovary removed (1,033, 1,034); to
both ovaries (1,041, 1,048) and to the left ovary
only (1,004). The heavy vertical line indicates
the irradiation date (60 days of age); m, date female was placed in male cage; rn-, date female
was removed from male cage. Sperm i n the
morning smear is indicated by a small vertical
line above the smear record. Number of implantation sites in the right and left uterine horns is
shown by numbers separated by a dash. Individual numbers indicate the number of young found
a t birth.
?
I
1
-
IMPLANTING OVA AFTER X-RAY
was even more marked. In these females,
only five of 12 animals produced implantations and no positive matings occurred
after 31 days following irradiation.
TABLE 1
Matings following irradiation of ovaries
800 r
to
No. of
animals
Fertile
matmgs
Sterile
matings
Both
Left R. out
22
22
12
50
27
5
9
98
ovary
Left
53
When only the left ovary was exposed
the average number of implantations per
pregnancy was rather constant (table 2).
As the number of implantations on the
irradiated side (left) decreased, there was
a corresponding increase in the productivity of the normal ovary. There was, however, a period of three to four weeks following irradiation in which the productivity of the irradiated ovary was comparable
to that of an untreated ovary. In ten
pregnancies of this period the left ovary
produced 54 implantations in contrast to
the 63 produced by the normal ovary. The
irradiated ovary in five of these animals
produced as many implantations, or more,
than the normal ovary. Furthermore, one
animal (1,002) accepted the male two days
after irradiation and two others (1,003
and 1,004) mated at three days. These
three females had been in estrus two days
prior to irradiation and the irradiated
ovaries produced a total of 15 ova, which
implanted, at the subsequent ovulation.
Whatever the damage or other effects of
irradiation on the maturation of the ovum
are (Harvey and Chang, '62; Mandl, '59c;
Spalding et al., '52; Russell and Russell,
'56 and others) the irradiation of these
three ovaries did not alter the estrous
cycles nor the number of viable ova produced.
There was a marked decrease in the
number of implantations on the left side
after the three to four week period. This
was also observed in subsequent pregnancies. However, ova were implanted on
the irradiated side as late as 213 days folfollowing irradiation. The decreased fertility of the irradiated ovary may have
been due to a decrease in the number of
259
ova as suggested by Ingram ('58). On the
other hand, Plagge and Krehbiel ('56)
noted that the irradiated ovary did not respond as well to gonadotrophins supplied
by a castrate parabiont in the presence of
the normal ovary. Also, Fels ('61) noted
that the damaged ovary reacted differently in the presence of a normal ovary.
This suggests a threshold difference in the
ovarian response to gonadotrophins after
the effects of unilateral irradiation become
apparent.
During the early post irradiation period
a normal number of implantations also
occurred after bilateral irradiation. Thereafter, these ovaries had a marked decrease
in productivity. There were instances,
however, of a single ovary producing a
normal complement of implanting ova at
relatively late dates following irradiation.
Examples were : four implantations on
days 93 and 145 (1,041), five on day 201
( 1,039) seven on days 75 (1,040) and
134 (1,039). In most of these instances
the opposite irradiated ovary failed to produce an implantation. This may have
been due to the gonadotrophic stimulus
being directed to but one ovary, or a given
dose of irradiation may not be uniform
in its effects, with one ovary being damaged more than the other. This cannot,
however, account for an animal (1,039)
in which all implantations of the second
pregnancy were on the right side and those
of the third pregnancy on the left side.
Also, the left ovary of another female
( 1,041) produced two implantating ova
at 145 days after failing to contribute to
earlier pregnancies. The present evidence
does not provide an adequate explanation
for these variations in ovarian activity.
When only one ovary produced implantations after bilateral irradiation a compensatory increase in numbers was not
generally observed. This was not due to
the length of the post irradiation period
since the average number of implantations
was 4.8 in five such pregancies during the
first 30 days after irradiation. The second
ovary may have ovulated ova which did
not implant. On the other hand, even if
not productive, this ovary may have utilized its share of the gonadotrophins. The
decrease in response to gonadotrophins by
irradiated ovaries (Ingram and Mandl, '58)
260
ROBERT H. KREHBIEL AND JAMES C. PLAGGE
TABLE
2
Implantations per pregnancy after irradiation of ovaries
Pregnancy number
2
1
No.
Days
post
r
Imp.
in
R. L.
Days
post
r
4
3
Imp.
in
R. L.
Days
post
r
Imp.
in
R. L.
5
Days
post
r
Imp.
R. inL.
Days
r
post
R.in L.
Imp.
-
~
800 T to left ovary
1002
1003
1004'
1001
1007
1006
1009
1005
1016
1008
1012
865
866
1018
1023
1019
1025
1021
1020
894
855
2
3
3
11
12
16
18
21
21
23
27
31
34
38
47
63
72
74
84
145
171
7
9
7
6
5
4
6
6
9
4
5
9
10
5
8
8
8
9
11
10
7
153
AvJpreg.
1060
1057
1059
1063
1061
1064
1056
1058
1041
867
1042
1048
1039
1040
1044
1043
7
8
12
12
15
15
17
22
23
32
34
41
47
75
76
77
5
6
4
0
7
7
6
7
3
9
2
3
3
2
0
0
0
0
2
1
0
67
10.5
7 8
6
4
5
0
0
5
2
7
7
4
8
6
7
3
1
72
Av./preg.
9
4
0
4
3
0
5
0
6
5
5
5
1
0
0
55
7.9
38
44
8 4
11 2
81
81
12 0
72
134
123
13 0
9 2
201
154
80
8 0
46
68
70
87
80
69
78
88
84
80
94
10
6
10
14
11
5
2
130
12 0
213
72
154
4 0
138
1
0
0
0
0
2
0
0
0
0
95
119
141
92
9 0
71
123
8 0
123
11 0
10 0
201
81
7
10
14
7
11
135 11
10.4
71 6
9.6
41
800 r to both ovaries
4 2
45
1
45
0 6
96
02
56
93
4 0
4 0
117
145
1 0
42
80
134
2 1
7 0
201
0 5
38 2
10.0
23 2
8.3
1
22 10
4.6
59
3.5
800 T to left w a r y - right ovary removed
1027
1068
1029
1031
1033
Av./preg.
8
10
11
20
31
7
10
4
9
12
42
8.4
1 Had sixth pregnancy at 205 days with 11-1, and a seventh pregnancy at 236 days with 9-0 implantations
(see fig. 1).
IMPLANTING OVA AFTER X-RAY
and the decrease in the number of eggs in
the ovary (Ingram, '58) should also be
considered. When both ovaries produced
during this period, however, the number
of implantations was normal.
In contrast to the extended period of
fertility seen occasionally in bilaterally
irradiated females, fertility in rats with
irradiated left ovary and with the right
ovary removed was limited to a very short
period. No pregnancies could be initiated
in this group after 31 days post irradiation.
The average number of implantations was
8.4. This number is significantly greater
( p < 0.02) than the number of implantations produced by the irradiated ovary in
the presence of a normal or a n irradiated
ovary during a comparable post irradiation
period. It is evident that the irradiated
ovary during this period can compensate
by producing more ova if the other ovary
has been removed. There is no evident
explanation for the failure of these animals to implant at a later date.
Resorptions were noted at the time of
exploration. No attempt was made to
determine the time or the cause of these.
The single daily observation of the isolated
female did not give an accurate count of
the number of young born. There was,
however, attrition of some magnitude between the implantation and birth periods.
An occasional prolonged pregnancy, terminating with a difficult labor, was noted.
LITERATURE CITED
Drips, D. G., and F. A. Ford 1932 The study
of the effects of roentgen rays on the estrual
cycle and the ovaries of the white rat. Surg.
Gyn. and Obst., 55: 596-606.
Fels, E. 1961 La ligadura unilateral del pediculo ovarico. Rev. SOC.Argentina Biol., 37:
4447.
26 1
Harvey, E. B., and M. C. Chang 1962 Effects
of x-irradation of ovarian ova of the golden
hamster. Anat. Rec., 142: 239.
Ingram, D. L. 1958 Fertility and oocyte numbers after x-irradiation of the ovary. J. Endocrin., 17: 61-90.
Ingram, D. L., and Anita M. Mandl 1958 The
hypophysial control of the x-ray sterilized
ovary. Ibid., 17: 1-12.
Krehbiel, R. H., and J. C. Plagge 1962 Distribution of ova in the rat uterus. Anat. Rec.,
143: 239-242.
Mandl, Anita M. 1959a Mating behaviour of
the adult rat after x-ray sterilization. J. Endocrin., 18: 434437.
195913 The oestrous cycle of the adult
rat after x-ray sterilization. Ibid., 18: 426-433.
1959c A quantitative study of the sensitivity of oocytes to x-irradiation. Proc. Roy.
SOC.B, 150: 53-71.
Nuzhdin, N. I., N. I. Shapiro, 0. N. Petrova and
0. N. Kitaeva 1955 Effect of ionizing radiations on the fertility of mice and the viability
of their progeny. C o d . Acad. Sci., USSR on
the peaceful uses of atomic energy. U. S.
Atomic Energy Com. Washington 1956, 4:
21-31.
Pencharz, R. I., and J. A. Long 1932 The effect
on a subsequent pregancy after x-raying one
ovary of a rat. Amer. J. Anat., 50: 1-8.
Plagge, J. C., and R. H. Krehbiel 1956 Histology of irradiated ovaries before and after
stimulation by gonadotrophins from a castrate
parabiont rat. Anat. Rec., 124: 348.
1962 Vaginal cycles in the rat after xray or other damage to one or both ovaries.
Ibid., 144: 287-293.
Roberts, Rough and Joan Wolff 1957 Threshold x-irradiation sterilization of the ovary. Fert.
Steril., 8: 428437.
Russell, Liane B., and W. L. Russell 1956 The
sensitivity of different stages i n oogenesis to
the radiation induction of dominant lethals
and other changes in the mouse. Progress i n
radiobiology. Mitchell, Holmes and Smith.
Edinburgh, Oliver and Boyd, 187-195.
Spalding, J. F., J. M. Wellnitz and W. H.
Schweitzer 1957 The effects of high dosage
x-ray on the maturation of the rat ovum and
their modification by gonadotrophins. Fert.
Steril., 8: 80-88.